This invention relates generally to products and processes useful to deliver proteins, including protein antigens, to the surface of gram-positive bacteria and firmly attach them to the cell. More specifically, it relates to the production of a fusion protein containing at least the anchor region of the cell associated region of a gram-positive surface protein and any protein, peptide or polypeptide that may be usefully administered to an animal host. The products of the invention which comprise proteins, peptides or polypeptides, as further discussed below, placed on the surface of a gram positive bacteria may be used to deliver such materials to the animal host to elicit an immungenic response, for example the production of protective antibodies or for another useful purpose. The protein, peptide or polypeptide may, for example, be an enzyme or other functional protein necessary for a specific purpose. It may be an antigenic determinant from a bacteria, virus, parasite or fungus. It may be a surface antigen from a mammalian tumor cell or of male sperm. It may be an allergen such as a vespid venom. The invention also relates to novel plasmids, genes, chromosomes and transformed bacteria employed in the production of such fusion proteins. The invention, additionally, provides novel vaccines which employ gram-positive bacteria designed to deliver to an animal host a foreign antigen normally present on a pathogenic microorganism which is associated with the virulence of that pathogen and which will elicit antigens to protect the host against infection or disease caused by the pathogenic microorganism.
The products of the invention are also useful as diagnostic agents.
The invention also provides a means to deliver enzymes, placed on the bacterial surface, to specific areas of interest.
The term xe2x80x9canimalxe2x80x9d as used herein refers to living beings including mammals such as man; bovines, especially beef cattle; sheep and goats; poultry, especially chickens, ducks and turkeys; as well as fish, especially those raised in fish farms such as salmon, trout and catfish. This invention is of special importance to mammals.
The essence of this invention is that it provides a method for the production of novel non-pathogenic gram positive bacteria expressing a hybrid surface protein which may be a hybrid surface antigen, comprising two principal parts, an anchor segment comprised of amino acid residues and an N-terminal active polypeptide segment, both of which will be defined and discussed in more detail below.
This invention will be better understood by consideration of the M protein and its structure. The M protein is a coiled coil surface antigen which is the virulence factor of group A streptococci, a gram positive bacteria. The M protein of Streptococcus pyogenes of M type 6 contains 441 amino acid residues.
Its structure will be discussed in more detail below, but it will be useful to discuss the cell associated region at this point. Using the standard one letter representation of amino acids, the structure of the anchor region of the cell associated region of the M6 protein from amino acid residue 407 to residue 441 may be represented as:
LPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN (SEQ ID NO:1)
Reading from the first leucine residue (L) at the N-terminal of the anchor region, the region includes the LPSTGE segment (SEQ ID NO:36); a spacer segment containing three amino acid residues TAN (SEQ ID NO:60); a hydrophobic segment of twenty amino acids, PFTAAALTVMATAGVAVV (SEQ ID NO:2); followed by a highly charged tail segment, KRKEEN (SEQ ID NO:61).
It has been observed as a result of structural studies of a large number of surface proteins of gram positive bacteria that the above described anchor region of the M6 protein is highly conserved among all known surface proteins of gram positive bacteria. Many of them are shown by Fischetti et al (Reference 1). Generally, the hydrophobic segment contains about 15 to 20 amino acid residues, the charged tail segment about 4 to 6 amino acid residues, and the spacer segment from about 3 to 6 amino acid residues. Most remarkable however, is the high degree of homology, practically 100% in the LPSTGE (SEQ ID NO:36) segment of the known surface proteins. The variations that occur are almost exclusively at the 3 and 6 positions. Therefore, the region may be generally represented as LPXTGX (SEQ ID NO:37).
The following Table 1 shows the remarkable extent of this homology thus far established amongst forty different surface proteins of gram positive bacteria.
It is apparent that this highly homologous region of the surface proteins of gram-positive bacteria is essential to anchoring bacterial surface proteins to the cell (28). This segment, which is referred to herein as the LPXTGX segment (SEQ ID NO: 37), is the crucial segment of the cell associated region of surface protein for anchoring the proteins to the surface of gram positive bacteria.
This discovery has been confirmed by Schneewind et al (65). Using the Protein A molecule of Staphylococcus aureus, these investigators have established that the complete complex (LPXTGX motif (SEQ ID NO:37), hydrophobic domain and charged tail) are necessary to deliver the Protein A molecule to the cell surface and that changes in the LPXTGX motif (SEQ ID NO:37) or deletion thereof will not inhibit expression of the molecule but will prevent it from anchoring to the surface.